Polyphenolic Extract of Euphorbia supina Attenuates Manganese-Induced Neurotoxicity by Enhancing Antioxidant Activity through Regulation of ER Stress and ER Stress-Mediated Apoptosis

Abstract

Manganese (Mn) is an important trace element present in human body, which acts as an enzyme co-factor or activator in various metabolic reactions. While essential in trace amounts, excess levels of Mn in human brain can produce neurotoxicity, including idiopathic Parkinson's disease (PD)-like extrapyramidal manganism symptoms. This study aimed to investigate the protective role of polyphenolic extract of Euphorbia supina (PPEES) on Mn-induced neurotoxicity and the underlying mechanism in human neuroblastoma SKNMC cells and Sprague-Dawley (SD) male rat brain. PPEES possessed significant amount of total phenolic and flavonoid contents. PPEES also showed significant antioxidant activity in 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and reducing power capacity (RPC) assays. Our results showed that Mn treatment significantly reduced cell viability and increased lactate dehydrogenase (LDH) level, which was attenuated by PPEES pretreatment at 100 and 200 µg/mL. Additionally, PPEES pretreatment markedly attenuated Mn-induced antioxidant status alteration by resolving the ROS, MDA and GSH levels and SOD and CAT activities. PPEES pretreatment also significantly attenuated Mn-induced mitochondrial membrane potential (ΔΨm) and apoptosis. Meanwhile, PPEES pretreatment significantly reversed the Mn-induced alteration in the GRP78, GADD34, XBP-1, CHOP, Bcl-2, Bax and caspase-3 activities. Furthermore, administration of PPEES (100 and 200 mg/kg) to Mn exposed rats showed improvement of histopathological alteration in comparison to Mn-treated rats. Moreover, administration of PPEES to Mn exposed rats showed significant reduction of 8-OHdG and Bax immunoreactivity. The results suggest that PPEES treatment reduces Mn-induced oxidative stress and neuronal cell loss in SKNMC cells and in the rat brain. Therefore, PPEES may be considered as potential treat-ment in Mn-intoxicated patients.

Keywords: Euphorbia supina; antioxidant; manganese; neuroprotection; neurotoxicity.

Figure 1

Protective effect of PPEES on Mn -induced cytotoxicity in SKNMC cell lines: (A) cell viability; and (B) LDH activity. Values were represented as mean ± SD (n = 3). ## p < 0.01 as compared with the control group; * p < 0.05; ** p < 0.01 as compared with the Mn alone group.

Figure 2

Protective Effect of PPEES on Mn-induced oxidative stress in SKNMC cell lines: (A) ROS; (B) malondialdehyde (MDA) levels; (C) superoxide dismutase (SOD) activity; (D) catalase (CAT) activity; and (E) glutathione (GSH) levels. Values were represented as mean ± SD (n = 3). ## p < 0.01 as compared with the control group; * p < 0.05 and ** p < 0.01 as compared with the Mn alone group.

Figure 3

The protective effect of PPEES against Mn-induced mitochondrial dysfunction in SKNMC cells. JC-1 fluorescent dye was used to measure the loss of mitochondrial membrane potential (ΔΨ_m). Values were represented as mean ± SD (n = 3). ## p < 0.01, compared to the control group; * p < 0.05; ** p < 0.01, compared to the Mn alone group.

Figure 4

Protective effects of PPEES against Mn-induced apoptosis in SKNMC cells: (A) representative pictures showing the apoptotic cells (Hoechst-positive cells) in arrowheads; and (B) representative percentage of the apoptotic rate, measured by calculating the percent of Hoechst positive cells over the total number of cells. Values were represented as mean ± SD (n = 3). ## p < 0.01, compared to the control group; * p < 0.05; ** p < 0.01, compared to the Mn alone group.

Figure 5

(A,B) Western blotting was performed to examine the effect of PPEES on the increased protein expression of GRP78, GADD34 and cleaved caspase-3, induced by administration of MnCl₂ (500 µM). Protein expression was normalized against β-actin. Values were represented as mean ± SD (n = 3). ## p < 0.01, compared to the control group; * p < 0.05; ** p < 0.01, compared to the Mn group.

Figure 6

(A–D) RT-PCR was performed to examine the effect of PPEES on the mRNA expression of XBP-1, CHOP, Bcl-2 and Bax in SKNMC cell that result from Mn treatment. GAPDH served as an internal control. The transcriptive levels of XBP1-1, CHOP, Bcl-2 and Bax were normalized against GAPDH. Values were represented as mean ± SD (n = 3). ## p < 0.01, compared to the control group; * p < 0.05; ** p < 0.01, compared to the Mn group.

Figure 7

The locomotors activity of normal control, Mn control and PPEES-treated rats was evaluated by open field OF test: (A) representation of time spent in the center of the arena; and (B) representation of number of squares traveled. Values were represented as mean ± SD (n = 5). ## p < 0.01 (normal control versus Mn-exposed rats), * p < 0.05, ** p < 0.01 (Mn-exposed versus Mn + PPEES rats).

Figure 8

Histopathological images showing the beneficial effects of PPEES on Mn-induced changes in rat striatum: (A) control group (Normal saline); (B) manganese chloride (15 mg/kg) treated group; (C) manganese chloride (15 mg/kg) + PPEES (100 mg/kg); and (D) manganese chloride (15 mg/kg) + PPEES (200 mg/kg) treated group (magnification at 10×). Damage (d); Ghost cells (g); hemorrhage (h); and vacuolated cytoplasm (s) (magnification at 10×).

Figure 9

IHC staining showing the protective effect of PPEES against Mn-induced neurodegenetive disease by reducing oxidized RNA in neurons. PPEES treatment significantly reduced 8-hydroxy-2′-deoxyguanosine (8-OHdG) expression that result from Mn exposure in striatum: (A) control group (Normal saline); (B) manganese chloride (15 mg/kg) treated group; (C) manganese chloride (15 mg/kg) + PPEES (100 mg/kg); and (D) manganese chloride (15 mg/kg) + PPEES (200 mg/kg) treated group (magnification at 40×).

Figure 10

Photographs showing protective effect of PPEES against Mn-induced neurotoxicity by reducing Bax expression in cortex of rats: (A) control group (Normal saline); (B) manganese chloride (15 mg/kg) treated group; (C) manganese chloride (15 mg/kg) + PPEES (100 mg/kg); and (D) manganese chloride (15 mg/kg) + PPEES (200 mg/kg) treated group (magnification at 40×).

Figure 11

The proposed mechanism of PPEES against Mn-induced toxicity. The schematic diagram shows Mn could exceed ROS; subsequently, altering activity of SOD and CAT. Changing GSH, MDA and 8-OHdG levels led to ER stress, followed by apoptosis through mitochondrial dysfunction. This diagram shows that PPEES prevents the Mn-induced neurotoxicity through regulation of ER stress and ER stress-mediated apoptosis.